Multi-drug resistance (MDR) in Gram-negative pathogens, including the Enterobacteriaceae and Pseudomonas aeruginosa, poses a significant threat to our ability to effectively treat infections caused by these organisms. A major component in the development of the MDR phenotype in Gram-negative bacteria is overexpression of RND-type efflux pumps, which actively pump antibacterial agents and biocides from the periplasm to the outside of the cell. Clearly, bacterial efflux pumps are an important target for developing novel antibacterial treatments that increase the potency of existing antibiotics and decrease the emergence of MDR bacteria. In preliminary studies, we identified a novel pyranopyridine (MBX2319) that is a potent inhibitor of AcrAB-TolC, the major efflux pump of E. coli and other Enterobacteriaceae. MBX2319 enhances the activity of fluoroquinolones (FQs) and -lactam antibiotics against E. coli, but does not exhibit antibacterial activity alone nor is it cytotoxic.In Phase I we synthesized MBX2319 analogs and evaluated them for potency, selectivity, spectrum of activity, and in vitro ADME properties to identify compounds with improved activity and drug-like properties, and to generate a molecular activity map for this series. As a result of this research, we have identified analogs that exhibit a >20-fold increase in antibiotic potentiation and satisfy the criteria for successful completion of the Phase I milestones. The overall goal of this Phase II project is to further develop the pyranopyridine series to identify 23 in vivo validated lead compounds that are suitable for IND-enabling preclinical studies. In Phase II, we will utilize an approach that combines structure based drug design with medicinal chemistry to design and synthesize analogs with improved spectrum of activity and ADMET properties while maintaining potency against efflux by the Enterobacteriaceae. To facilitate this approach and probe the mechanism of action, we will generate a three dimensional structure of MBX2319 and analogs bound to AcrB. Analogs will be evaluated in a panel of secondary assays to prioritize compounds for efficacy and pharmacokinetic (PK) studies in animals. In addition, the data derived from these assays will inform the design of additional compounds. Through an iterative process of compound design and evaluation, we anticipate that we will identify 2-3 in vivo validated lead compounds with favorable PK and in vivo efficacy. In Phase III, these efflux pump inhibitors will be developed for use in combination with levofloxacin (LEV) or piperacillin/tazobactam (PIP/TAZ) as an adjunctive therapy for urinary tract and bloodstream infections as the first therapeutic indications for this inhibitor series. These adjunctive therapis represent a significant improvement over single agent therapies, because they will provide the following benefits: 1) increased antibiotic efficacy at lower concentrations, and 2) decreased evolution of resistance. The Specific Aims for Phase II are as follows. Aim 1. Chemically optimize the pyranopyridine series to generate lead compounds for animal safety and efficacy testing. Aim 2. Prioritize analogs by potency, spectrum, selectivity, favorable in vitro ADMET properties. Aim 3. Determine the three dimensional structure of pyranopyridines bound to AcrB and biochemical mechanism. Aim 4. Evaluate acute toxicity, pharmacokinetics, and efficacy of lead compounds in animal models.